Gram-positive bacteria, such as Staphylococci, Enterococci and Clostridia, are extremely important, pathogens in both human and veterinary medicine. In the United States, between 1995 and 1998, 60% of hospital bloodstream infections involved gram-positive bacteria. This percentage is continuing to increase. The development of antibiotic resistance amongst gram-positive bacteria complicates treatment and can lead to increased morbidity and mortality.
Antibiotic resistance in bacteria has been selected through the prolific use of these drugs both in human medicine and animal husbandry, indiscriminate prescribing practices, and patient non-compliance with treatment regimes. Therapeutic options for the treatment of such drug-resistant microorganisms, especially gram-positive bacteria, are becoming increasingly limited. The problem of antibiotic resistance is exacerbated by the spread of drug-resistant organisms, and the dissemination of resistance genes between bacteria. The threat to the successful management of bacterial infections posed by the development and spread of antibiotic resistance is one of the most significant problems within healthcare and veterinary medicine.
Staphylococci 
Staphylococci are major causes of serious healthcare associated infection (HAI). Of particular note are strains of Staphylococcus that have developed or obtained varying levels of resistance to antibiotics such as methicillin (meticillin). These difficult-to-treat organisms are commonly known as methicillin resistant Staphylococcus aureus (MRSA) and methicillin resistant Staphylococcus epidermidis (MRSE). Approximately 80% of S. epidermidis isolates from device-associated infections are methicillin resistant (MRSE) as well as being multi-resistant. Resistance to multiple antibiotics and the ability of S. epidermidis to form biofilms on inert surfaces exacerbate the challenges of treating infections caused by these organisms.
In the USA, over 50% of clinical S. aureus isolates are resistant to the β-lactam methicillin (NNIS, 2004). Similarly, reports of methicillin-resistant S. aureus (MRSA) in animals have become more frequent in recent years (O'Mahony et al., 2005); MRSA has been isolated from dogs, cats, cattle, sheep, chickens, rabbits and horses (Devriese and Hommez, 1975, Hartmann et al., 1997, Pak et al., 1999, Tomlin et al., 1999, Lee, 2003, Goni et al., 2004, and Weese 2004).
In both human and veterinary medicine, bacterial biofilms (structured communities of bacterial cells enclosed in a self-produced polymeric matrix and adherent to an inert or living surface (Costerton et al., 1999)) are a significant problem. In animal husbandry, bacterial biofilms can develop on poultry processing instrumentation (Arnold & Silvers, 2000) and may cause treatment failure of mastitis in cows infected with S. aureus (Melchior et al., 2006). In human healthcare, biofilms of bacteria have been shown to colonise many medical devices, including orthopaedic implants (Bahna et al., 2007). In the UK, 35% of hip prostheses' infection is attributable to S. aureus, resulting in septic loosening, fracture non-union and osteomyelitis (Sanderson, 1991). The association of MRSA with the use of orthopaedic devices is extremely problematic due to the increased spectrum of resistance of this organism, in addition to the protection from the immune system given by the biofilm growth phase, often necessitating the removal of a contaminated device, causing further trauma to the patient and increasing medical costs. Colonisation with MRSA is the general precursor to the development of an MRSA infection, so interventions that reduce levels of human colonisation or the colonisation of surfaces such as medical devices will reduce the spread of infections in healthcare facilities.
The acquisition of methicillin resistance among Staphylococcal species not only precludes the use of all currently available β-lactam antibiotics, but also is commonly associated with resistance to multiple drug classes.
Methicillin resistance in Staphylcocci develops by the alteration of the target of the drug. β-lactam antibiotics, such as methicillin, act on sensitive strains by binding to and inhibiting proteins called “Penicillin Binding Proteins”. Resistance to methicillin in Staphylococci occurs by the alteration one of these proteins, PBP2′, so that β-lactams bind poorly to it. This results in the bacterium becoming resistant to all currently available β-lactam drugs. MRSA and MRSE infections can be treated with glycopeptide drugs, such as vancomycin. The rise in prevalence of MRSA and MRSE, in addition to emerging high levels of resistance to aminoglycosides and ampicillin in Enterococci, have resulted in an increased reliance on vancomycin. This has driven the subsequent emergence of vancomycin resistant pathogens. Of particular note are strains commonly known as vancomycin intermediately sensitive Staphylococcus aureus (VISA) and vancomycin resistant Staphylococcus aureus (VRSA), all of which are multi-drug resistant and difficult to treat. The emergence of VISA and VRSA means that current antibiotics may become ineffective for the treatment of human infections such as endocarditis, bacteraemia and osteomyelitis.
The administration of vancomycin to patients with recurrent MRSA infections causes an increased risk of the emergence of VISA or VRSA strains. The vast majority of VISA infections in the USA occur in patients with recurrent MRSA treated with vancomycin (Appelbaum, 2006). Although a dramatic reduction in the use of glycopeptides such as vancomycin would reduce the emergence and spread of VISA and VRSA, this is not practical without the use of alternative compounds that do not promote the emergence of multiple resistance.
Clostridia 
Clostridia are multi-drug resistant Gram-positive bacteria that are becoming one of the most difficult to treat healthcare-associated infections to date. The administration of broad-spectrum antibiotics, such as ampicillin, amoxicillin and the cephalosporins, plays a key role in the development of Clostridium difficile-associated diarrhoea (CDAD). The presence of a large number of mobile genetic elements within the genome of C. difficile are thought to be responsible for the multiple-drug resistance observed in this species. The use of broad-spectrum antibiotics reduces bacterial colonisation in the intestine, permitting the overgrowth of C. difficile. Clostridium difficile is extremely hardy; by forming spores, it can survive extremes of temperature, ethanol and antibiotics, and so is very difficult to treat. One of the key challenges in the treatment of C. difficile is the fact that it is able to form these highly resistant spores. As such, antibiotic treatment commonly results in the inhibition of the actively growing Clostridium cells but not the vegetative spores. The spores remain in the gut of a mammal after treatment and can then germinate resulting in a new C. difficile infection. Currently the antibiotics of choice for the treatment of C. difficile are metronidazole and then vancomycin if metronidazole is ineffective. However, neither of these drugs is able to inhibit the outgrowth of Clostridium spores, and as such the prevalence of relapses in C. difficile infection after treatment is estimated at around 55% of all cases.
Initially, oral vancomycin was used as a primary drug choice in the treatment of CDAD, but because of the risk of promotion and selection of vancomycin resistant gut flora (such as Enterococci), vancomycin is recommended only for cases that do not respond to the primary treatment (metronidazole). Recently, resistance to vancomycin and metronidazole in C. difficile isolates has been reported, and the use of these agents has been shown to increase the density of vancomycin-resistant Enterococcus (VRE) in the stools of colonised patients.
In summary, there are three key factors to be considered when designing novel therapeutics agents for the treatment of Clostridia, these are:                1. The drug must be able to kill the multiply-resistant actively growing Clostridia to give efficient relief to the patient from the disease.        2. The drug must be able to prevent the outgrowth of Clostridium spores to minimise the likelihood of a relapse.        3. A drug that does not promote the spread of vancomycin resistance is preferable.Enterococci         
The development of vancomycin-resistant Enterococci (VRE) in recent years is of major significance. Enterococci once were viewed as harmless inhabitants of the human and animal gut flora, but have now acquired resistance to multiple classes of antibiotic, including the last-resort drug, vancomycin. In the US, the prevalence of Enterococcus faecium exhibiting vancomycin resistance rose from 26.2% in 1995 to around 70% in 2004, making it one of the most feared pathogens in US hospitals.
The acquisition of vancomycin resistance among some strains of Enterococci is associated with resistance to multiple drug classes due to the sequential nature at which these strains have acquired resistance to every new antibiotic challenge.
Genotypes and Phenotypes of Multi-Drug Resistant Gram Positives
MRSA, MRSE, VISA, VRSA and VRE are genotypically and phenotypically distinct from other, sensitive Staphylococci and Enterococci, tending to form discrete clonal lineages.
The most prevalent clones of MRSA in the UK are EMRSA-15 and EMRSA-16; EMRSA-16 is regarded as endemic in the majority of UK hospitals. These MRSA clones differ from other Staphylococci by the presence of a cassette of several genes (the SCCmec gene cassette), and are commonly resistant to many different classes of antibiotic in addition to methicillin. This gene cassette contains the genes for methicillin resistance as well as genes important for enabling the cassette to move between strains; it commonly contains many other genes encoding resistance to other antibiotic classes. A genetic comparison between an EMRSA-16 strain and a sensitive Staphylococcal strain revealed that the MRSA strain contained an extra 106 genes, many of which were important for the virulence and drug resistance of the strain.
Similarly, VRE and Clostridium outbreaks are commonly clonal, with the vanA gene cassette (coding for cell-wall precursors that do not bind to vancomycin) being the most prevalent genetic resistance mechanism in VRE outbreaks. In the UK it is estimated that only 3 clonal strains of C. difficile are responsible for around 75% of all CDAD cases.
Mechanisms of Drug Resistance
Drug resistance can be specific, i.e. particular to a certain drug or class of drugs, or non-specific in that the resistance applies to a range of drugs, not necessarily related. In the case of VISA, an increase in cell wall thickness is a major contributor to the observed drug resistance.
VISA and VRSA may be defined as any Staphylococcal strain with a vancomycin MIC of 4-8 mg/L (VISA) or greater or equal to 8 mg/L (VRSA). These levels of resistance can be due to an increase in cell wall thickness, by the production of cell-wall precursors incapable of binding vancomycin, or via another mechanism. Susceptible gram-positive organisms synthesise cell wall precursors ending in D-ala-D-ala, whereas vancomycin resistant gram-positive organisms, such as VISA, VRSA and VRE synthesise, for example, D-ala-D-lac precursors. The presence of vancomycin resistance in Staphylococcal and Enterococcal strains may be identified by the measurement of the MIC to vancomycin by broth or agar dilution, or by Etest®, or by the identification of vanA, vanB, vanC, vanD, vanE, vanG genes, or similar, by polymerase chain reaction (PCR). The current invention also encompasses the subclass of VISA strains that are heterogeneous VISA (hVISA); these are vancomycin susceptible methicillin-resistant Staphylococcus aureus by conventional testing but have a sub-population of intermediately resistant cells. hVISA strains are thought to be the precursors of VISA.
Management of Multi-Drug Resistant Gram Positive Infections
The management of human infections caused by MRSA, MRSE, VISA, VRSA and VRE reflect the genotypic and phenotypic differences outlined above, and require greater investment in hospital infrastructure, facilities for patient isolation, and infection control measures than for other strains of Staphylococci and Enterococci. The ease at which Clostridium difficile can spread within the hospital environment, and the ability of the bacterium to form highly-resistant spores, means that C. difficile infections also require more extensive infection control measures than those required for most other gram-positive infections; recent cost estimates attributable to CDAD in the UK and USA exceed US$4,000 per case.
The treatment options for infections contributed to or caused by VISA, VRSA and VRE are now severely limited. Resistance against the two newest antibiotics for VRE (quinupristin-dalfopristin and linezolid) as been described; linezolid has already been associated with treatment failure in VRE infections. There is an urgent need to discover new compounds that inhibit or kill such organisms, and to limit the development and spread of these multiply-resistant pathogens.
Current treatment for Clostridium difficile is not always effective; there are increasing reports of recurring infection and resistance development. CDAD recurs after treatment in up to 55% of patients. Because of the limited therapeutic options for the treatment of CDAD and the high recurrence rate, new therapeutic strategies targeting both the growing bacterial cells and vegetative spores are urgently needed.
It has been found that certain imidazoles and or their derivatives are capable of inhibiting the growth of Clostridium difficile (George, 1979), MRSA (Lee & Kim, 1999) and/or VISA, VRSA and VRE. However, the identification of compounds that act synergistically with these drugs (the imidazoles) means that lower concentrations of original drug may be used (thus reducing the undesirable side effects of the imidazoles) and prolonging the life of the drug treatment (e.g. a synergistic combination of two drugs will require resistance to develop in both components before the combination becomes ineffective). If the spontaneous rate of resistance development in an organism is 108, the development of resistance to the combination of two compounds will be approximately 106, therefore the risk of resistance developing is dramatically lower.
Synergy between antibiotics may occur when two antibiotics target bacterial proteins within the same metabolic pathway. Trimethoprim and sulphamethoxazole are commonly administered together as co-trimoxazole because they target two different enzymes in the bacterial folic acid synthesis pathway. Synergy may also occur when a resistance mechanism, such as an efflux pump, is inhibited, permitting the accumulation of an antibiotic that if administered singly, may be removed by the efflux pump. There is no known technique by which to predict that two compounds will act synergistically to give an antibacterial effect greater than the sum of the effects of the individual drugs, unless the mechanism of action of each agent is known, and even then, synergy is not guaranteed. Similarly, if a compound acts synergistically with a particular antibiotic, it cannot be predicted that a combination with an antibiotic acting on different bacterial targets or on different bacterial strains will also exhibit synergy.
It is known that bacitracin and miconazole act synergistically against Staphylococcus aureus and Staphylococcus epidermidis (Cornelissen and Bossche, 1983). However, when this combination was tested against multi-drug resistant strains such as MRSA and VISA (this specification, Table 3), synergy did not occur. This highlights the fact that phenotypic and genotypic differences between sensitive and resistant strains prevents the prediction of synergy from data generated on sensitive strains. Additionally, not all cell wall or membrane active agents will demonstrate synergy with miconazole or other imidazoles when used to kill or inhibit the growth of bacteria.
Nisin is known to have in vitro activity against C. difficile (Bartoloni A et al, 2004; Kerr et al, 1997) and the potential for nisin and vancomycin to be synergistic is disclosed. However, as described above, this information does not allow predictions of synergy of nisin with other antibiotics, especially as there is a lack of clarity over the exact mode of action of miconazole against Clostridium. For example, nisin does not act synergistically with other antibiotics such as bacitracin and chloramphenicol, which like vancomycin also act on the cell wall (chloramphenicol causes an accumulation of cell wall peptidoglycan). Indeed, nisin was found to antagonise the antibacterial activity of chloramphenicol, thus, one cannot predict that synergy will occur with nisin and miconazole.
Similarly, fosfomycin, and derivatives thereof, is known to be active against some sensitive Gram positive bacteria, and synergy has been shown with some antibiotics (such as rifampicin and linezolid) but not with other antibiotics (e.g. vancomycin) (Grif et al, 2001). This demonstrates that the detection of synergy with some antibiotics cannot be used to generate blanket predictions of synergy with other drugs.
The present invention discloses the knowledge that the combination of certain imidazoles with one or more specific agents active on a bacterial cell membrane or bacterial cell wall, is capable of inhibiting the growth of MRSA, MRSE, VISA, VRSA, VRE and Clostridia at dramatically lower concentrations than either agent used singly, or their additive effect. The combination of miconazole with nisin demonstrates surprising synergy against the actively growing cells, but an additional benefit of this combination is that the nisin component also acts on the vegetative cells by inhibiting their outgrowth, thus reducing the likelihood of a relapse in infection.
An objective of the present invention is to provide a new and effective treatment for infections contributed to or caused by difficult to treat gram-positive bacteria, such as, MRSA, MRSE, VISA, VRSA, VRE and Clostridia. 